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R&D Systems hspb5
Hspb5, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 3 article reviews

hspb5 - by Bioz Stars, 2026-03

93/100 stars

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Construction of HSPB5-BioID2 expression plasmid and BioID activity. ( a ) Schematic diagram of BioID2 fusion protein. ( b ) Subcellular localization of HSPB5-BioID2 expression. HeLa cells were transfected with expression plasmid, and 24 h after addition of biotin (50 μM), cells were immunostained with anti-HA antibody and stained with fluorescently labeled secondary antibody, streptavidin-Alexa Fluor 546, and with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI). White arrows indicate lamellipodia around cell membrane. Scale bar indicates 20 μm. ( c ) Detection of BioID2 fusion protein expression and BioID activity. 293T cells were transfected with expression plasmid, and cells were collected 24 h after addition of biotin. Expression of BioID2 fusion protein was detected with an anti-HA antibody. ( d ) Biotinylated proteins were detected using streptavidin−peroxidase polymer. Arrowheads (HSPB1-BioID2) and arrows (HSPB5-BioID2) indicate positions of respective proteins. HSPB1 is an isoform that belongs to the same sHsp family as HSPB5.

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: Construction of HSPB5-BioID2 expression plasmid and BioID activity. ( a ) Schematic diagram of BioID2 fusion protein. ( b ) Subcellular localization of HSPB5-BioID2 expression. HeLa cells were transfected with expression plasmid, and 24 h after addition of biotin (50 μM), cells were immunostained with anti-HA antibody and stained with fluorescently labeled secondary antibody, streptavidin-Alexa Fluor 546, and with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI). White arrows indicate lamellipodia around cell membrane. Scale bar indicates 20 μm. ( c ) Detection of BioID2 fusion protein expression and BioID activity. 293T cells were transfected with expression plasmid, and cells were collected 24 h after addition of biotin. Expression of BioID2 fusion protein was detected with an anti-HA antibody. ( d ) Biotinylated proteins were detected using streptavidin−peroxidase polymer. Arrowheads (HSPB1-BioID2) and arrows (HSPB5-BioID2) indicate positions of respective proteins. HSPB1 is an isoform that belongs to the same sHsp family as HSPB5.

Article Snippet: The following antibodies were used for immunostaining or immunofluorescence applications: HSPB5 (NBP1-47708, Novus Biologicals, Centennial, CO, USA), pHSPB5 (pSer19; C7740, Sigma-Aldrich Japan KK, Tokyo, Japan), pHSPB5 (pSer45; NB120-5598, Novus), pHSPB5 (pSer59; ab5577, Abcam, KK, Tokyo, Japan), β-tubulin (014-25041, Fujifilm Wako Pure Chemicals, Osaka, Japan), p38 (612168, BD Transduction Laboratories, Franklin Lakes CO, USA), and pp38 (pThr180/pTyr182; 612280, BD).

Techniques: Expressing, Plasmid Preparation, Activity Assay, Transfection, Staining, Labeling, Membrane, Polymer

List of candidate binding proteins for HSPB5 identified by DiDBit method.

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: List of candidate binding proteins for HSPB5 identified by DiDBit method.

Techniques: Binding Assay

PLK2, a novel binding protein of HSPB5, is induced by MG132 in L6 cells. ( a ) Schematic diagram of domain structure of PLK2. K90, K95, and K111 indicate position of lysine residue (K) biotinylated by HSPB5-BioID2. Lysine residues were numbered based on amino acid sequence of PLK2 derived from Homo sapiens (NCBI NP_006613). ( b ) Comparison of induction of HSPB5 and PLK2 expression by ER stress-induced drugs. Differentiated L6 cells were treated with 5 µM MG132 or 0.1 µg/mL tunicamycin for 24 h. Induction of HSPB5, and PLK2 protein expression was detected by Western blotting. ( c ) Graph shows levels of endogenous proteins induced by the drug. Data represent means ± standard error (n = 3). Statistical analyses were performed using t -test. ** p < 0.01; n.s. means not significant. ( d ) Verification of binding of PLK2 to HSPB5. FLAG-HSPB5 was overexpressed in L6 cells by liposome transfection. After 5 µM MG132 treatment (24 h), cells were solubilized, and HSPB5 pull-down assay was performed using an anti-tag antibody. ( e ) Quantification of binding of PLK2 to HSPB5. Control cells were transfected with empty vector. After 5 µM MG132 treatment (24 h), a pull-down assay was performed using an anti-tag antibody. Graph presents level of endogenous PLK2 pulled down by HSPB5. Data represent means ± standard error (n = 3). Statistical analyses were performed using t -test. * p < 0.05.

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: PLK2, a novel binding protein of HSPB5, is induced by MG132 in L6 cells. ( a ) Schematic diagram of domain structure of PLK2. K90, K95, and K111 indicate position of lysine residue (K) biotinylated by HSPB5-BioID2. Lysine residues were numbered based on amino acid sequence of PLK2 derived from Homo sapiens (NCBI NP_006613). ( b ) Comparison of induction of HSPB5 and PLK2 expression by ER stress-induced drugs. Differentiated L6 cells were treated with 5 µM MG132 or 0.1 µg/mL tunicamycin for 24 h. Induction of HSPB5, and PLK2 protein expression was detected by Western blotting. ( c ) Graph shows levels of endogenous proteins induced by the drug. Data represent means ± standard error (n = 3). Statistical analyses were performed using t -test. ** p < 0.01; n.s. means not significant. ( d ) Verification of binding of PLK2 to HSPB5. FLAG-HSPB5 was overexpressed in L6 cells by liposome transfection. After 5 µM MG132 treatment (24 h), cells were solubilized, and HSPB5 pull-down assay was performed using an anti-tag antibody. ( e ) Quantification of binding of PLK2 to HSPB5. Control cells were transfected with empty vector. After 5 µM MG132 treatment (24 h), a pull-down assay was performed using an anti-tag antibody. Graph presents level of endogenous PLK2 pulled down by HSPB5. Data represent means ± standard error (n = 3). Statistical analyses were performed using t -test. * p < 0.05.

Techniques: Binding Assay, Residue, Sequencing, Derivative Assay, Comparison, Expressing, Western Blot, Transfection, Pull Down Assay, Control, Plasmid Preparation

MG132 induced ER stress in L6 cells. ( a ) Induction of ER stress by MG132. After differentiation (48 h), L6 myotubes were treated with 5 µM MG132. Induction of CHOP expression was detected by Western blotting. ( b ) Effect of MG132 treatment on cell viability and cytotoxicity of L6 cells. After differentiation, L6 myotubes were treated with 5 µM MG132 (24 h). Cytotoxicity was measured from activity of lactate dehydrogenase in the medium. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using t -test. ** p < 0.01. ( c ) Activation of stress-related kinases by MG132 treatment. After 5 µM MG132 treatment (24 h), phosphorylation of MK2 (pThe334) and p38 (pThr180/pTyr182) was detected by Western blotting. ( d ) Colocalization of endogenous HSPB5 and PLK2. After differentiation (48 h), endogenous HSPB5 and PLK2 were immunofluorescently stained and observed by confocal microscopy. BI2536 is a PLK-specific inhibitor. BI2536 (10 nM) was treated simultaneously with 5 µM MG132 for 24 h. Yellow arrows indicate cells with co-localization of HSPB5 and PLK2 in perinuclear ER region. Scale bar indicates 20 μm.

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: MG132 induced ER stress in L6 cells. ( a ) Induction of ER stress by MG132. After differentiation (48 h), L6 myotubes were treated with 5 µM MG132. Induction of CHOP expression was detected by Western blotting. ( b ) Effect of MG132 treatment on cell viability and cytotoxicity of L6 cells. After differentiation, L6 myotubes were treated with 5 µM MG132 (24 h). Cytotoxicity was measured from activity of lactate dehydrogenase in the medium. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using t -test. ** p < 0.01. ( c ) Activation of stress-related kinases by MG132 treatment. After 5 µM MG132 treatment (24 h), phosphorylation of MK2 (pThe334) and p38 (pThr180/pTyr182) was detected by Western blotting. ( d ) Colocalization of endogenous HSPB5 and PLK2. After differentiation (48 h), endogenous HSPB5 and PLK2 were immunofluorescently stained and observed by confocal microscopy. BI2536 is a PLK-specific inhibitor. BI2536 (10 nM) was treated simultaneously with 5 µM MG132 for 24 h. Yellow arrows indicate cells with co-localization of HSPB5 and PLK2 in perinuclear ER region. Scale bar indicates 20 μm.

Techniques: Expressing, Western Blot, Activity Assay, Activation Assay, Phospho-proteomics, Staining, Confocal Microscopy

PLK2 phosphorylates HSPB5 at serine 19 under ER stress. ( a ) Knockdown efficiency of PLK2 by siRNA. L6 cells were transfected with siRNA to knockdown PLK2 and treated with 5 µM MG132 for 24 h. Graph presents relative value of expression level of PLK2. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using t -test. ** p < 0.01. ( b ) Schematic representation of three known phosphorylation sites of HSPB5. Arrows indicate name of the kinase catalyzing phosphorylation. ( c ) Effect of PLK2 on each phosphorylation site. Undifferentiated L6 cells were transfected with siRNA to knockdown PLK2. After differentiation (48 h), L6 myotubes were treated with 5 µM MG132 for 24 h. Phosphorylation level was detected by Western blotting with specific antibodies. Graph presents ratio of phosphorylated HSPB5/HSPB5 after quantification of each band. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey test. ‡ p < 0.01, † p < 0.05; n.s. not significant. ( d ) Effect of PLK2 activity on phosphorylation of serine 19. After differentiation (48 h), L6 myotubes were treated with 10 nM BI2536 and 5 µM MG132 for 24 h. Phosphorylation level was detected by Western blotting with specific antibodies.

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: PLK2 phosphorylates HSPB5 at serine 19 under ER stress. ( a ) Knockdown efficiency of PLK2 by siRNA. L6 cells were transfected with siRNA to knockdown PLK2 and treated with 5 µM MG132 for 24 h. Graph presents relative value of expression level of PLK2. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using t -test. ** p < 0.01. ( b ) Schematic representation of three known phosphorylation sites of HSPB5. Arrows indicate name of the kinase catalyzing phosphorylation. ( c ) Effect of PLK2 on each phosphorylation site. Undifferentiated L6 cells were transfected with siRNA to knockdown PLK2. After differentiation (48 h), L6 myotubes were treated with 5 µM MG132 for 24 h. Phosphorylation level was detected by Western blotting with specific antibodies. Graph presents ratio of phosphorylated HSPB5/HSPB5 after quantification of each band. Data in graphs represent means ± standard error (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey test. ‡ p < 0.01, † p < 0.05; n.s. not significant. ( d ) Effect of PLK2 activity on phosphorylation of serine 19. After differentiation (48 h), L6 myotubes were treated with 10 nM BI2536 and 5 µM MG132 for 24 h. Phosphorylation level was detected by Western blotting with specific antibodies.

Techniques: Knockdown, Transfection, Expressing, Phospho-proteomics, Western Blot, Activity Assay

Relationship between phosphorylation and subcellular localization of HSPB5. ( a ) Localization of overexpressed recombinant HSPB5 and endogenous PLK2. After 5 µM MG132 treatment (24 h), FLAG-tagged HSPB5 and endogenous PLK2 in undifferentiated L6 cells were immunofluorescently stained with anti-tag and anti-PLK2 antibodies. ( b ) Effect of PLK2 on localization of HSPB5 and desmin protein. Endogenous HSPB5 and desmin were immunofluorescently stained with anti-HSPB5 and anti-desmin antibodies. Undifferentiated L6 cells were transfected with siRNA to knockdown PLK2. After differentiation (48 h), L6 myotubes were treated with MG132 for 24 h. White arrowheads indicate cells with colocalization of HSPB5 and desmin at perinuclear ER region. Scale bar indicates 20 μm.

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: Relationship between phosphorylation and subcellular localization of HSPB5. ( a ) Localization of overexpressed recombinant HSPB5 and endogenous PLK2. After 5 µM MG132 treatment (24 h), FLAG-tagged HSPB5 and endogenous PLK2 in undifferentiated L6 cells were immunofluorescently stained with anti-tag and anti-PLK2 antibodies. ( b ) Effect of PLK2 on localization of HSPB5 and desmin protein. Endogenous HSPB5 and desmin were immunofluorescently stained with anti-HSPB5 and anti-desmin antibodies. Undifferentiated L6 cells were transfected with siRNA to knockdown PLK2. After differentiation (48 h), L6 myotubes were treated with MG132 for 24 h. White arrowheads indicate cells with colocalization of HSPB5 and desmin at perinuclear ER region. Scale bar indicates 20 μm.

Techniques: Phospho-proteomics, Recombinant, Staining, Transfection, Knockdown

HSPB5 is involved in inhibition of caspase 3 via phosphorylation. ( a ) Knockdown efficiency of HSPB5 by siRNA. L6 cells were transfected with siRNA to knockdown HSPB5 and treated with 5 µM MG132 for 24 h. Graph presents relative value of expression level of HSPB5. ( b ) Effect of HSPB5 on ER stress-induced caspase 3 activity. Undifferentiated L6 cells were transfected with siRNA by lipofection. After differentiation (48 h), L6 myotubes were treated with MG132 for 24 h. Undifferentiated L6 cells were transfected with siRNA to knockdown HSPB5. After differentiation, L6 myotubes were treated with 5 µM MG132 for 24 h. Caspase 3 activity was quantified from ratio of procaspase 3 to cleaved caspase 3 (cleaved caspase 3/procaspase 3) by Western blotting using specific antibodies. Graph shows ratio of cleaved caspase 3/procaspase 3 after quantifying each band, relative toMG132 untreated siRNA control. ( c ) Effect of PLK2 on ER stress-induced PARP activity. Undifferentiated L6 cells were transfected with siRNA by lipofection. After differentiation (48 h), L6 myotubes were treated with 5 µM MG132 for 24 h. Undifferentiated L6 cells were transfected with siRNA to knockdown HSPB5. After differentiation (48 h), L6 myotubes were treated with MG132 for 24 h. PARP activity was quantified from ratio of PARP to cleaved PARP by Western blotting. Graph shows cleaved PARP/PARP ratio after quantifying each band relative to MG132 untreated siRNA control. ( d ) Inhibitory effect of HSPB5 protein on ER stress-induced caspase 3 activity. Undifferentiated L6 cells were transfected with expression plasmid by lipofection. Control cells were transfected with empty expression vector. After differentiation, L6 myotubes were treated with 5 µM MG132 for 24 h. Graph shows ratio of cleaved caspase 3/procaspase 3 after quantifying each band, relative to MG132 untreated control. Data in graphs represent mean ± standard error (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey test or t -test. ** p < 0.01 ( t -test). ‡ p < 0.01, † p < 0.05; n.s. means not significant (Tukey test).

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: HSPB5 is involved in inhibition of caspase 3 via phosphorylation. ( a ) Knockdown efficiency of HSPB5 by siRNA. L6 cells were transfected with siRNA to knockdown HSPB5 and treated with 5 µM MG132 for 24 h. Graph presents relative value of expression level of HSPB5. ( b ) Effect of HSPB5 on ER stress-induced caspase 3 activity. Undifferentiated L6 cells were transfected with siRNA by lipofection. After differentiation (48 h), L6 myotubes were treated with MG132 for 24 h. Undifferentiated L6 cells were transfected with siRNA to knockdown HSPB5. After differentiation, L6 myotubes were treated with 5 µM MG132 for 24 h. Caspase 3 activity was quantified from ratio of procaspase 3 to cleaved caspase 3 (cleaved caspase 3/procaspase 3) by Western blotting using specific antibodies. Graph shows ratio of cleaved caspase 3/procaspase 3 after quantifying each band, relative toMG132 untreated siRNA control. ( c ) Effect of PLK2 on ER stress-induced PARP activity. Undifferentiated L6 cells were transfected with siRNA by lipofection. After differentiation (48 h), L6 myotubes were treated with 5 µM MG132 for 24 h. Undifferentiated L6 cells were transfected with siRNA to knockdown HSPB5. After differentiation (48 h), L6 myotubes were treated with MG132 for 24 h. PARP activity was quantified from ratio of PARP to cleaved PARP by Western blotting. Graph shows cleaved PARP/PARP ratio after quantifying each band relative to MG132 untreated siRNA control. ( d ) Inhibitory effect of HSPB5 protein on ER stress-induced caspase 3 activity. Undifferentiated L6 cells were transfected with expression plasmid by lipofection. Control cells were transfected with empty expression vector. After differentiation, L6 myotubes were treated with 5 µM MG132 for 24 h. Graph shows ratio of cleaved caspase 3/procaspase 3 after quantifying each band, relative to MG132 untreated control. Data in graphs represent mean ± standard error (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey test or t -test. ** p < 0.01 ( t -test). ‡ p < 0.01, † p < 0.05; n.s. means not significant (Tukey test).

Techniques: Inhibition, Phospho-proteomics, Knockdown, Transfection, Expressing, Activity Assay, Western Blot, Control, Plasmid Preparation

A novel PLK2/HSPB5 pathway is proposed as a model for cytoprotection against ER stress by MG132. PLK2, a novel binding protein of HSPB5, phosphorylates serine 19 (pSer19) of HSPB5 under ER stress conditions caused by proteome inhibitor MG132. This phosphorylation is responsible for inhibitory effect of HSPB5 on ER stress-induced apoptosis by MG132. PLK2/HSPB5 pathway provides a molecular mechanism for pSer19 of HSPB5 and represents part of mechanism for suppressing ER stress-induced apoptosis via proteasome inhibition.

Journal: International Journal of Molecular Sciences

Article Title: Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5

doi: 10.3390/ijms231911257

Figure Lengend Snippet: A novel PLK2/HSPB5 pathway is proposed as a model for cytoprotection against ER stress by MG132. PLK2, a novel binding protein of HSPB5, phosphorylates serine 19 (pSer19) of HSPB5 under ER stress conditions caused by proteome inhibitor MG132. This phosphorylation is responsible for inhibitory effect of HSPB5 on ER stress-induced apoptosis by MG132. PLK2/HSPB5 pathway provides a molecular mechanism for pSer19 of HSPB5 and represents part of mechanism for suppressing ER stress-induced apoptosis via proteasome inhibition.

Techniques: Binding Assay, Phospho-proteomics, Inhibition

Transcript levels were measured for hspb2, hspb1, hspb5, hspb6 in HSPB2wt and HSPB2cKO animals, after sham or TAC-surgery. The transcript abundance increased significantly in response to pressure-overload. Except for hspb2, there were no significant differences between sHsp mRNA expression in HSPB2wt and HSPB2cKO hearts in both of sham and TAC operated mice. From 3 to 5 heart samples were analyzed per group. *P<0.05compared with each sham groups.

Journal: PLoS ONE

Article Title: HSPB2 Is Dispensable for the Cardiac Hypertrophic Response but Reduces Mitochondrial Energetics following Pressure Overload In Mice

doi: 10.1371/journal.pone.0042118

Figure Lengend Snippet: Transcript levels were measured for hspb2, hspb1, hspb5, hspb6 in HSPB2wt and HSPB2cKO animals, after sham or TAC-surgery. The transcript abundance increased significantly in response to pressure-overload. Except for hspb2, there were no significant differences between sHsp mRNA expression in HSPB2wt and HSPB2cKO hearts in both of sham and TAC operated mice. From 3 to 5 heart samples were analyzed per group. *P<0.05compared with each sham groups.

Article Snippet: The proteins were transferred to nitrocellulose membrane and were probed with antibodies against HSPB2 (see description above), CRYAB/HSPB5 antibody (1∶5000) , HSP20 (HSPB6 (ADI-SPA-796, Enzo Life Sciences; 1∶2000), HSP25/HSPB1 (ADi-SPA- 801, Enzo Life Sciences; 1∶1000), VDAC (porin, PC548, Calbiochem: 1∶1000) and GAPDH (GAPDH -14C10, Cell signaling technology; 1∶5000).

Techniques: Expressing

$Hearts from HSPB2wt and HSPB2cKO were isolated at 8 weeks after sham or TAC procedure and protein extracts (soluble fraction) were analyzed by western blots probed with anti-HSPB2, HSPB1 (HSP25), HSPB5 (CRYAB) and HSPB6 (HSP20). In contrast to transcripts, protein levels were not significantly modified in samples from TAC operated mice and except for HSPB2, there was no difference between HSPB2cKO and HSPB2wt samples. Two to five samples were analyzed per group and a representative example is shown.$

Journal: PLoS ONE

Article Title: HSPB2 Is Dispensable for the Cardiac Hypertrophic Response but Reduces Mitochondrial Energetics following Pressure Overload In Mice

doi: 10.1371/journal.pone.0042118

Figure Lengend Snippet: Hearts from HSPB2wt and HSPB2cKO were isolated at 8 weeks after sham or TAC procedure and protein extracts (soluble fraction) were analyzed by western blots probed with anti-HSPB2, HSPB1 (HSP25), HSPB5 (CRYAB) and HSPB6 (HSP20). In contrast to transcripts, protein levels were not significantly modified in samples from TAC operated mice and except for HSPB2, there was no difference between HSPB2cKO and HSPB2wt samples. Two to five samples were analyzed per group and a representative example is shown.

Techniques: Isolation, Western Blot, Modification

Journal: Bioengineered

Article Title: The crystallin alpha B (HSPB5)-tripartite motif containing 33 (TRIM33) axis mediates myocardial fibrosis induced by angiotensinogen II through transforming growth factor-β (TGF-β1)-Smad3/4 signaling

doi: 10.1080/21655979.2022.2054913

Figure Lengend Snippet: qRT-PCR primer sequences

Article Snippet: The primary antibodies applied in the study included TRIM33 (CST, 90051S, diluted 1:1000), HSPB5 (CST, 45844S, diluted 1:1000), tubulin (CST, 2148S, diluted 1:1000), TGF-β1 (CST, 84912S, diluted 1:1000), α-SMA (CST, 19245S, diluted 1:1000), β-catenin (CST, 8480S, diluted 1:1000), FOXO3a (CST, 12829S, diluted 1:1000), Col 1 (CST, 72026S, diluted 1:1000), p-Smad3 (CST, 9520S, diluted 1:1000), Smad3 (CST, 9523S, diluted 1:1000), p-Smad4 (Invitrogen, PA5-12,695, diluted 1:1000), Smad4 (CST, 46535S, diluted 1:1000), and the internal reference GAPDH (CST, 5174S, diluted 1:1000).

Techniques:

HSPB5-TRIM33 axis suppresses TGF-β1-Smad3/4 signaling in CFs. a-d. Western blot analysis was used to detect the expression of TGF-β1, β-catenin and FOXO3a in CFs after TRIM33 overexpression. e-g. The P-smad3/Smad3 and P-smad4/Smad4 level after TRIM33 overexpression was determined by Western blot. h-j. Western blot analysis was used to detect the expression of HSPB5 and TRIM33 in HSPB5 overexpressed CFs. Each experiment was conducted three times independently. **P < 0.01, ***P < 0.001, ns: No statistical significance.

Journal: Bioengineered

doi: 10.1080/21655979.2022.2054913

Figure Lengend Snippet: HSPB5-TRIM33 axis suppresses TGF-β1-Smad3/4 signaling in CFs. a-d. Western blot analysis was used to detect the expression of TGF-β1, β-catenin and FOXO3a in CFs after TRIM33 overexpression. e-g. The P-smad3/Smad3 and P-smad4/Smad4 level after TRIM33 overexpression was determined by Western blot. h-j. Western blot analysis was used to detect the expression of HSPB5 and TRIM33 in HSPB5 overexpressed CFs. Each experiment was conducted three times independently. **P < 0.01, ***P < 0.001, ns: No statistical significance.

Techniques: Western Blot, Expressing, Over Expression

TRIM33 attenuates the fibrosis induced by HSPB5. a. The qRT-PCR analysis was used to clarify the expression of HSPB5. b-c. EDU immunofluorescence staining was conducted to detect the proliferation rate of CFs. d-e. The migration assay was used to confirm the migration of CFs in four groups. f-g. The Coll I and a-SMA expression level was determined by Western blot. Each experiment was conducted three times independently. *P < 0.05,**P < 0.01, ***P < 0.001.

Journal: Bioengineered

doi: 10.1080/21655979.2022.2054913

Figure Lengend Snippet: TRIM33 attenuates the fibrosis induced by HSPB5. a. The qRT-PCR analysis was used to clarify the expression of HSPB5. b-c. EDU immunofluorescence staining was conducted to detect the proliferation rate of CFs. d-e. The migration assay was used to confirm the migration of CFs in four groups. f-g. The Coll I and a-SMA expression level was determined by Western blot. Each experiment was conducted three times independently. *P < 0.05,**P < 0.01, ***P < 0.001.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Migration, Western Blot

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